Effect of O2-monobutyryl guanosine 3', 5'-cyclic monophosphate on hepatic metabolism of free fatty acids.

Livers from fed male rats were perfused $\text{in vitro}$ with O^2′-monobutyryl guanosine 3′,5′-cyclic monophosphate. The output of triglyceride was reduced, while output of ketone bodies and glucose was stimulated by 10^?4M monobutyryl guanosine 3′,5′-cyclic monophosphate. No effect was observed with 10^?5 M nucleotide. Monobutyryl guanosine 3′,5′-cyclic monophosphate did not affect uptake of free fatty acids. In these respects, monobutyryl guanosine 3′,5′-cyclic monophosphate mimics the effects of dibutyryl adenosine 3′,5′-cyclic monophosphate, although the guanylic nucleotide seems to be less potent than the adenosine 3′,5′-cyclic monophosphate derivative.     

Cyclic nucleotide esterification: relationship to phosphate ring geometry and growth regulation in synchronized human lymphocytes.

Infrared spectra of neutral aqueous solutions of nucleoside 3′,5′-cyclic monophosphates indicate an increase in the antisymmetric phosphoryl stretching frequency to 1236 cm^?1 from 1215 cm^?1 in trimethylene cyclic phosphates. A further increase to 1242 cm^?1 accompanies esterification of the 2′-ribose hydroxyl. The O²′-esterified and 2′-deoxy cyclic nucleotides examined display both reduced kinase binding and altered phosphoryl stretching frequencies, suggesting that modification of the phosphate ring represents a common feature in decreased kinase activation. Reversible inhibition of mitosis in thymidine-synchronized human lymphocytes by 2 mmN⁶,O²′-dibutyryladenosine 3′,5′-cyclic monophosphate and N⁶-monobutyryladenosine 3′,5′-cyclic monophosphate was observed. However, adenosine 3′,5′-cyclic monophosphate, O²′-monobutyryladenosine 3′,5′-cyclic monophosphate, butyric acid, and ethyl butyrate had no effect on mitosis when present at 2 mm concentrations during S and G2. These results are consistent with hydrolysis of O²′-monobutyryladenosine 3′,5′-cyclic monophosphate and adenosine 3′,5′-cyclic monophosphate by esterase and phosphodiesterase enzymes and suggest that modification of the N⁶ amino group is necessary for the antimitotic activity of N⁶,O²′-dibutyryladenosine 3′, 5′-cyclic monophosphate.     

Oxidation Chemistry of Adenosine-3′, 5′-Cyclic Monophosphate at Pyrolytic Graphite Eletrode

The voltammetric oxidation of adenosine-3′,5′-cyclic monophosphate (3′,5′-CAMP) has been studied in the pH range 2.13–10.07 using pyrolytic graphite electrode (PGE). Voltammetric, coulometric, spectral studies, and product characterization indicate that the oxidation of 3′,5′-CAMP occurs in an EC reaction involving a 6H⁺, 6e process at pH 7.24. Electrooxidized products were seperated by semipreparative high performance liquid chromatography (HPLC) and were characterized by mp, ¹HNMR, FTIR, and GC-mass as allantoin cyclic ribose monophosphate and 3 dimers as the major products. A detailed interpretation of the redox mechanism of 3′,5′-CAMP also has been presented to account for the formation of various products.     

Hydrolysis of 2',3'-cyclic adenosine monophosphate and 3',5'-cyclic adenosine monophosphate in subcellular fractions of normal and neoplastic mouse spleen

A comparison has been made between the capacity to hydrolyse 2′,3′-cyclic adenosine monophosphate and 3′,5′-cyclic adenosine monophosphate in subcellular fractions of normal and neoplastic (lymphosarcoma) spleen of C₅₇BL mice. The effect of X-irradiation on these activities was tested. Subcellular fractionation of normal and lymphosarcoma spleen points to a different overall localization of the enzymes. The 2′,3′-cyclic nucleotide phosphodiesterase (2′,3′-cAMPase) has its highest specific activity in the particulate fractions of the cell, while the data on 3′,5′-cyclic nucleotide phosphodiesterase (3′,5′-cAMPase) show the highest activity in the soluble fraction. The 2′,3′-cAMPase activity is higher in the tumor as compared to the normal tissue, while the opposite holds for 3′,5′-cAMPase. Total body irradiation of normal mice with a dose of 600 rads of X-rays, results in a clear drop in 2′,3′-cAMPase 48 hours after the exposure. The 3′,5′-cAMPase is hardly affected at this time. Neither imidazol nor Mg⁺⁺ has any influence on the 2′,3′-cAMPase. The pH optimum for 3′,5′-cAMPase and 2′,3′-cAMPase appears to be 7.7 and 6.2 respectively. This report suggests a no-identity of the two enzymes in mouse spleen, a situation different from that found in certain plants.     

Variation in isomeric products of a phosphodiesterase from the chloroplasts of Phaseolus vulgaris in response to cations

ABSTRACT

Fast-atom bombardment mass spectrometry (FABMS), and collisionally-induced dissociation and mass-analyzed ion kinetic energy spectrum scanning (CID/MIKES) have been used to examine cation effects on a Phaseolus chloroplast complex phosphodiesterase activity. The kinetic parameters of the activity, and the effects of Li⁺, Na⁺, K⁺, Mg²⁺, Mn²⁺ and Fe³⁺ upon them, were determined with 3′,5′-cyclic AMP, -GMP and -CMP, and 2′,3′-cyclic AMP, -GMP and -CMP as substrates. Irrespective of the presence of cations and of the complex nucleotidase, the preferred substrate is a 3′,5′-cyclic nucleotide, not a 2′,3′-cyclic nucleotide. In the presence of the nucleotidase 3′,5′-cyclic AMP and 3′,5′-cyclic GMP are the best substrates, unless Fe³⁺ ions are present. Mg²⁺ and Mn²⁺ stimulate hydrolysis of 3′,5′-cyclic AMP and 3′,5′-cyclic GMP by the complex. However, Fe³⁺ inhibits these activities but stimulates the hydrolysis of 3′,5′-cyclic CMP. Kinetic data indicate that each of these six substrates is hydrolyzed at a single, common, catalytic site. Differentiation of the phosphodiesterase isomeric mononucleotide products by FABMS CID/MIKES analysis indicates that in the absence of ions and after removal of the nucleotidase, the 3′-ester linkage of the 3′,5′-cyclic substrates was hydrolyzed exclusively. Addition of monovalent and divalent ions results in hydrolysis of both the 5′- and 3′-ester linkages.     

Adenosine 3' , 5'-cyclic monophsphate phosphodiesterase activities in the x-irradiation induced rat small bowel adenocarcinoma.

The adenosine 3′, 5′-cyclic monophosphate phosphodiesterase (PDE) activities were evaluated in X-irradiation induced Holtzman rat small bowell adenocarcinoma and age-matched normal small intestine. Within normal small intestine, PDE activity was optimal at pH 7.4, and highly dependent upon the addition of Mg²⁺ or Mn²⁺. Analyses of the rat small bowel adenocarcinoma revealed significantly elevated PDE activities above the normal small bowel which were found to be relatively constant throughout the length of the ileum and jejunum. These findings suggest that the diminished intracellular adenosine 3′, 5′-cyclic monophosphate levels observed in this lesion (1) may be the consequence of elevated PDE activities.     

Synthesis and Antitumor Properties of Some Neutral Triesters of 5-Fluoro-2′-deoxyuridine-5′-monophosphate and 3′,5′-Cyclic Monophosphate

Abstract

Several new prodrugs of 5-fluoro-2′-deoxyuridine 5′-monophosphate and 3′,5′-cyclic monophosphate were synthesized and their antitumor activities were evaluated in vitro.     

Nitric oxide and downstream second messenger cGMP and cAMP enhance adipogenesis in primary human preadipocytes

Background aimsObesity is correlated with chronic low-grade inflammation. Thus the induction of inflammation could be used to stimulate adipose tissue formation in tissue-engineering approaches. As nitric oxide (NO) is a key regulator of inflammation, we investigated the effect of NO and its downstream signaling molecule guanosine 3′,5′-cyclic monophosphate (cGMP) as well as adenosine 3′,5′-cyclic monophosphate (cAMP) on preadipocytes in vitro.MethodsPreadipocytes were isolated from human subcutaneous adipose tissue, cultured until confluence, and differentiated. The NO donor diethylenetriamine (DETA)/NO (30–150 μm) was added during proliferation and differentiation. Additionally, cGMP/cAMP analogs 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP), 8-(4-chlorophenylthio)-guanosine 3′,5′-cyclic monophosphate (8-pCPT-cGMP) and 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP), and the adenylyl cyclase activator forskolin, specific guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and adenylyl cyclase inhibitor 2′-5′-dideoxyadenosine (ddA), were applied. Proliferation and differentiation were evaluated.ResultsDETA/NO in combination with the standard differentiation procedure significantly enhanced maturation of precursor cells to adipocytes. Proliferation, in contrast, was inhibited in the presence of NO. The application of cGMP and cAMP, respectively, increased pre-adipocyte differentiation to an even higher extent than NO. Inhibitors of the underlying pathways caused a significant decrease in adipogenic conversion.ConclusionsOur results support the application of NO donors during transplantation of preadipocytes in a 3-dimensional setting to accelerate and optimize differentiation. The results suggest that, instead of the rather instable and reactive molecule NO, the application of cGMP and cAMP would be even more effective because these substances have a stronger adipogenic effect on preadipocytes and a longer half-life than NO. Also, by applying inhibitors of the underlying pathways, the induced inflammatory condition could be regulated to the desired level.     

Partial characterization of 5′(3′)-ribonucleotide and 3′-ribonucleotide phosphohydrolases from Tradescantia albiflora leaves

Two acid phosphomonoesterases, 5′(3′)-ribonucleotide phosphohydrolase and 3′-ribonucleotide phosphohydrolase, were isolated from Tradescantia albiflora leaf tissue and purified by ammonium sulphate precipitation, gel filtration on Sephadex G-200 and repeated chromatography on DEAE-cellulose. The enzymes differed in their sensitivity to dialysis against 1 mM EDTA; the activity of 5′(3′)-ribonucleotide phosphohydrolase was unaffected, while 3′-ribonucleotide phosphohydrolase showed an increase of 60–90%. Both enzymes were rapidly inactivated above 50°. Their ion sensitivity was identical: 1 m M Zn²⁺ and Fe²⁺ were inhibitors for both by 20–80%; while Mg²⁺, Ca²⁺, Co²⁺, K⁺, Na⁺ at 1–10 mM had no significant effect on the activity of either enzyme. Inorganic phosphate inhibited both enzymes almost completely. EDTA (1 mM) did not inhibit either enzyme; none of the divalent cations tested were enzyme activators. 3′-Ribonucleotide phosphohydrolase hydrolysed both 3′- and 5′-nucleoside monophosphates (3′-AMP, 3′-CMP, 3′-GMP, 3′-UMP, 5′-AMP, 5′-CMP, 5′-GMP, 5′-UMP). 5′(3′)-Ribonucleotide phosphohydrolase showed a preference for the 3′-nucleoside monophosphates. Adenosine 3′,5′-cyclic monophosphate, purine and pyrimidine 2′,3′-cyclic mononucleotides at 0.1–1.OmM did not inhibit the enzymes.     

Identification of cytidine 2′,3′-cyclic monophosphate and uridine 2′,3′-cyclic monophosphate in Pseudomonas fluorescens pfo-1 culture

Cytidine 2′,3′-cyclic monophosphate (2′,3′-cCMP) and uridine 2′,3′-cyclic monophosphate (2′,3′-cUMP) were isolated from Pseudomonas fluorescens pfo-1 cell extracts by semi-preparative reverse phase HPLC. The structures of the two compounds were confirmed by NMR and mass spectroscopy against commercially available authentic samples. Concentrations of both intracellular and extracellular 2′,3′-cCMP and 2′,3′-cUMP were determined. Addition of 2′,3′-cCMP and 2′,3′-cUMP to P. fluorescens pfo-1 culture did not significantly affect the level of biofilm formation in static liquid cultures.     

Proton magnetic resonance studies of 9-(β-d-xylofuranosyl)adenine 3′,5′-cyclic monophosphate and 9-(β-d-arabinofuranosyl)adenine 2′,5′-cyclic monophosphate

A detailed ¹H 220-MHz n.m.r. study of 9-(β-d-xylofuranosyl)adenine 3′,5′-cyclic monophosphate (3′,5′-xylo-cAMP, 1) and 9-(?-d-arabinofuranosyl)adenine 2′,5′-cyclic monophosphate (2′,5′-ara-cAMP, 2) in D₂O solution is described. The sugar-ring conformations in 1 and 2 are shown to be ³E and ²E, respectively, and the phosphate rings are in a chair form. An unusual ⁴J_P,H coupling of 2.4 Hz is observed between H-4′ and phosphorus in 1 and a vicinal J_P,H of 30.8 Hz between H-5′ and phosphorus in 2. This latter coupling verifies a similar value found previously in the ara-cytidine analog of 2. A comparison of the conformational properties of cyclic nucleotides having fused phosphate and sugar rings has been made, together with an assessment of the use of the Karplus constants in such ring-systems.     

CHANGE IN THE ACTIVITY OF LIPASE IN SEA URCHIN EGGS AND EMBRYOS DURING EARLY DEVELOPMENT*

During the early development of the sea urchin, Anthocidaris crassispina, the activity of lipase was maintained at the same level as in unfertilized eggs until the mesenchymal blastula stage (20 hr culture at 20°C) and then increased gradually after gastrulation. The activity in the embryos kept in SO^2?₄-free artificial sea water changed in a similar manner to that in those kept in normal sea water, during the development until 36 hr of fertilization. At 48 hr, the activity in the embryos, which had developed to the permanent blastulae in SO^2?₄-free sea water, was markedly lower than in normal plutei and was similar to that in unfertilized eggs. The lipase activity in fertilized eggs 30 min after fertilization, which was almost the same as that in unfertilized eggs was found mainly to be localized in the precipitate fraction obtained by the centrifugation at 12,000 x g for 20 min, whereas the activity in unfertilized eggs was found in the precipitate by the centrifugation at 105,000 x g for 60 min. Ca²⁺, adenosine 3′, 5′-cyclic monophosphate (cAMP) and guanosine 3′, 5′-cyclic monophosphate (cGMP) had no effect on the lipase activity.     

The effect of N6-2'-O-dibutyryl-adenosine 3',5'-monophosphate on rat liver calciferol 25-hydroxylase.

Liver calciferol 25-hydroxylase activity of vitamin-D deficient rats was enhanced 24 hours following the intravenous injection of N⁶-2′-O-dibutyryl adenosine 3′,5′-monophosphate. Sodium butyrate administered in the same way had no effect on this enzyme system. Administration of actinomycin D with N⁶-2′-O-dibutyryl adenosine 3′,5′-monophosphate abolished the stimulatory effect of the cyclic nucleotide. Direct addition to the incubation medium of adenosine 3′,5′-cyclic monophosphate or of its dibutyryl derivative did not influence the hepatic conversion of cholecalciferol to 25-hydroxycholecalciferol. These results suggest a possible role for the cyclic nucleotide in the regulation of this enzyme system.     

Specific localization of 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca2+/Mg2+)-ATPase,and acetylcholinesterase in human erythyrocyte membrane

A procedure was developed for the detection of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in myelin. This assay was sufficiently sensitive to detect the low levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in human erythrocytes. The 2′,3′-cyclic nucleotide 3′-phosphohydrolase of human erythrocytes was determined to be exclusively associated with the inner (cytosolic) side of the membrane. Leaky ghostsand resealed ghosts were assayed for 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca²⁺/Mg²⁺-ATPase, and acetylcholinesterase activity, and the 2′,3′-cyclic nucleotide 3′-phosphohydrolase profile is the same as that of the (Ca²⁺/Mg²⁺)-ATPase, an established inner membrane maker.     

Neurotrophic control of cyclic nucleotide levels during muscle differentiation in cell culture

The effects of chick brain–spinal cord extract on morphological development and cyclic nucleotide levels of cultured chick embryo skeletal muscle cells were determined. It had previously been shown that the extract stimulated morphological differentation, protein synthesis, and cholinesterase activity of muscle cells. Myoblasts fused earlier and an increase in number as well as diameter of myotubes were seen in the extract treated cultures. Cyclic nucleotides levels were higher (almost twice the controls for both adenosine 3′, 5′ -cyclic monophosphate and guanosine 3′, 5′ -cyclic monophosphate) and preceded their occurence in the control cultures. It was suggested that factor(s) in the extract interact with membrane receptor(s) to alter nucleotide levels which, in turn, allow the effects to be expressed.     

Synthesis and Deprotection of Biodegradably and Thermally Protected Dinucleoside‐2′,5′‐Monophosphate Prodrug Model of 2‐5A

Protected dinucleoside‐2′,5′‐monophosphate has been prepared to develop a prodrug strategy for 2‐5A. The removal of enzymatically and thermally labile 4‐(acetylthio)‐2‐(ethoxycarbonyl)‐3‐oxo‐2‐methylbutyl phosphate protecting group and enzymatically labile 3′‐O‐pivaloyloxymethyl group was followed at pH 7.5 and 37 °C by HPLC from the fully protected dimeric adenosine‐2′,5′‐monophosphate 1 used as a model compound for 2‐5A. The desired unprotected 2′,3′‐O‐isopropylideneadenosine‐2′,5′‐monophosphate ( 9 ) was observed to accumulate as a major product. Neither the competitive isomerization of 2′,5′‐ to a 3′,5′‐linkage nor the P–O5′ bond cleavage was detected. The phosphate protecting group was removed faster than the 3′‐O‐protection and, hence, the attack of the neighbouring 3′‐OH on phosphotriester moiety did not take place.     

Purification and Some Properties of Adenosine (Phosphate) Deaminase from the Liver of the Squid Todarodes pacificus

An enzyme that catalyzed the deamination of adenosine 3′-phenylphosphonate was purified from squid liver to homogeneity as judged by SDS-PAGE. The molecular weight of the enzyme was estimated to be 60,000 by SDS-PAGE and 140,000 by Sephadex G-150 gel filtration. The enzyme deaminated adenosine, 2′-deoxyadenosine, 3′-AMP, and 2′,3′-cyclic AMP, but not adenine, 5′-AMP, 3′,5′-cyclic AMP, ADP, or ATP. The apparent K_m and V_max at pH 4.0 for these substrates were comparable (0.11-0.34mM and 179-295 μmol min^?1 mg^?1, respectively). The enzyme had maximum activity at pH 3.5-4.0 for adenosine 3′-phenylphosphonate, at pH 5.5 for adenosine and 2′-deoxyadenosine, and at pH 4.0 for 2′,3′-cyclic AMP and 3′-AMP when the compounds were at concentration of 0.1 mM. The K_m at 4.0 and 5.5 for each substrate varied, but the Vmax were invariant. These results indicated that the squid enzyme was a novel adenosine (phosphate) deaminase with a unique substrate specificity.     

In silico design of novel hERG-neutral sildenafil-like PDE5 inhibitors

Cyclic nucleotide phosphodiesterase enzymes (PDEs) have functions in regulating the levels of intracellular second messengers, 3′, 5′-cyclic adenosine monophosphate (cAMP) and 3′, 5′-cyclic guanosine monophosphate (cGMP), via hydrolysis and decomposing mechanisms in cells. They take essential roles in modulating various cellular activities such as memory and smooth muscle functions. PDE type 5 (PDE5) inhibitors enhance the vasodilatory effects of cGMP in the corpus cavernosum and they are used to treat erectile dysfunction. Patch clamp experiments showed that the IC₅₀ values of the human ether-à-go-go-related gene (hERG1) potassium (K) ion channel blocking affinity of PDE5 inhibitors sildenafil, vardenafil, and tadalafil as 33, 12, and 100 μM, respectively. hERG1 channel is responsible for the regulation of the action potential of human ventricular myocyte by contributing the rapid component of delayed rectifier K⁺ current (I_Kr) component of the cardiac action potential. In this work, interaction patterns and binding affinity predictions of selected PDE5 inhibitors against the hERG1 channel are studied. It is attempted to develop PDE5 inhibitor analogs with lower binding affinity to hERG1 ion channel while keeping their pharmacological activity against their principal target PDE5 using in silico methods. Based on detailed analyses of docking poses and predicted interaction energies, novel analogs of PDE5 inhibitors with lower predicted binding affinity to hERG1 channels without loosing their principal target activity were proposed. Moreover, molecular dynamics (MD) simulations and post-processing MD analyses (i.e. Molecular Mechanics/Generalized Born Surface Area calculations) were performed. Detailed analysis of molecular simulations helped us to better understand the PDE5 inhibitor–target binding interactions in the atomic level. Results of this study can be useful for designing of novel and safe PDE5 inhibitors with enhanced activity and other tailored properties.     

Improved synthesis of 32P-labelled 3′,5′-cyclic AMP, 3′,5′-cyclic GMP and other 3′,5′cyclic ribo- and deoxyribonucletodies of high specific activity

A general procedure is described for the two-step chemical synthesis from [³²P]orthophosphoric acid of the eight common ribo- and deoxyribonucleoside 3′,5′-cyclic monophosphates. The method is simple and reliable and both steps are carried out in the same reaction flask without an intermediate purification step. ³²P-labelled cyclic nucleotides are obtained after paper chromatography in yields of 20–60% relative to starting [³²P]orthophosphoric acid and with a specific activity of greater than 1 mCi/μmole. Alternative methods for the purification of reaction mixtures and for the preparation of ³²P-labelled 3′,5′-cyclic AMP and 3,′,5′-cyclic GMP are described.     

Identification and characterization of the adenosine 3′,5′-cyclic monophosphate binding proteins appearing during the development of Dictyostelium discoideum

A photosensitive, radioactive analogue of cyclic adenosine monophosphate, 8-azido-adenosine 3′,5′-[³²P]monophosphate (8-N₃-cyclic AMP), was used to label the cyclic AMP binding proteins of Dictyostelium discoideum. During development cytosolic proteins appear which are specifically labeled by the photoaffinity agent. The proteins are developmentally regulated since they are only found in starved, developing cells. Unlabeled cyclic AMP competes specifically with the labeled analogue for protein binding sites in contrast to unlabeled 5′-AMP which does not compete. A mutant which develops spores but is deficient in stalk cell production produces a different set of cyclic AMP binding proteins from the parent strain.